Sequences for FK228 biosynthesis and methods of synthesizing FK228 and FK228 analogs
Polynucleotides encoding the polypeptides involved in biosynthesis of FK 228 and those involved in synthesis of a novel FK228 analog, thailandepsin are disclosed herein. Also provided are methods of making FK228, thailandepsin and analogs of these molecules and methods of using these FK228 analogs. Chromobacterium and Burkholderia gene inactivation mutants are provided. Methods of forming a disulfide bond in a chemical are also disclosed.
1. A method of making FK228 comprising growing a recombinant cell comprising exogenous polynucleotides encoding proteins DepA of SEQ ID NO: 29, DepB of SEQ ID NO: 31, DepC of SEQ ID NO: 33, DepD of SEQ ID NO: 35, DepE of SEQ ID NO: 37, DepF of SEQ ID NO: 39, DepH of SEQ ID NO: 43, and DepM of SEQ ID NO: 25, the polynucleotides operably connected to a promoter, under conditions that allow synthesis of FK228.
2. The method of claim 1 , wherein the cell is a bacterium of a genus selected from the group consisting of Chromobacterium, Pseudomonas, Escherichia, Salmonella, Burkholderia, Bifidobacterium , or Clostridium.
3. The method of claim 1 , further comprising introducing a polynucleotide encoding protein DepL of SEQ ID NO: 23.
4. The method of claim 1 wherein the polynucleotides comprise depA of SEQ ID NO: 28, depB of SEQ ID NO: 30, depC of SEQ ID NO: 32, depD of SEQ ID NO: 34, depE of SEQ ID NO: 36, depF of SEQ ID NO: 38, depH of SEQ ID NO: 42, and depM of SEQ ID NO: 24.
5. The method of claim 1 , further comprising introducing a polynucleotide sequence encoding protein DepG of SEQ ID NO: 41.
6. The method of claim 1 , further comprising introducing a polynucleotide sequence encoding protein DepI of SEQ ID NO: 45.
7. The method of claim 1 , further comprising introducing a polynucleotide sequence encoding protein DepJ of SEQ ID NO: 47.
8. The method of claim 1 , further comprising introducing a polynucleotide sequence encoding protein DepK of SEQ ID NO: 21.
9. The method of claim 1 , further comprising introducing at least one polynucleotide sequence encoding at least one protein selected from the group consisting of DepG of SEQ ID NO: 41, DepL of SEQ ID NO: 23, DepI of SEQ ID NO: 45, DepJ of SEQ ID NO: 47, and DepK of SEQ ID NO: 21, or any combination thereof.
10. The method of claim 1 wherein the polynucleotide comprises SEQ ID NO: 1.